Compounds and methods for inhibiting β-protein filament formation and neurotoxicity

ABSTRACT

Methods of treating an individual with Alzheimer&#39;s Disease by administering to the patient a therapeutically effective dose of a compound which interferes with the interaction between Alzheimer β-protein and Apolipoprotein E4 or α 1  -antichymotrypsin, thereby suppressing the formation of Alzheimer β-protein filaments and the neurotoxic effects of these filaments. The present invention also refers to methods of screening for compounds which are effective drugs for treating Alzheimer&#39;s disease. These methods comprise screening for compounds which suppress the formation of Alzheimer β-protein filaments in the presence of promoting factors and which suppress the neurotoxic effects of these filaments formed in the presence of promoting factors.

GOVERNMENT FUNDING

This invention was made with Government support under Contract No.AG08084, AG09665 and GM35967 awarded by NIH. The Government has certainrights in the invention.

RELATED APPLICATIONS

This application is a Continuation-in-Part of U.S. Ser. No. 08/328,491,filed Oct. 25, 1994, entitled Compounds and Methods for Inhibitingβ-Protein Filament Formation and Neurotoxicity, now abandoned, which isa Continuation-in-Part of U.S. Ser. No. 08/290,198, filed Aug. 15, 1994,entitled "Compounds and Methods for Inhibiting β-Protein FilamentFormation and Neurotoxicity," by Huntington Potter (abandoned), which isa Continuation-in-Part of U.S. application Ser. No. 08/179,574 filedJan. 10, 1994, entitled "Compounds and Methods for Inhibiting β-ProteinFunction," by Huntington Potter and Usamah Kayyali, U.S. Pat. No.5,506,097 which is a Continuation-in-Part of U.S. application Ser. No.07/819,361, filed Jan. 13, 1992, entitled "Method of Interfering withFormation of α₁ -Antichymotrypsin-β-Protein Complex, Method ofInhibiting β-Protein Function and Compounds for Use Therein," byHuntington Potter and Usamah Kayyali, U.S. Patent No. 5,338,663 which isa Continuation-in-Part of U.S. application Ser. No. 07/572,671, filedAug. 24, 1990, entitled "Method of Interfering with Formation of α₁-Antichymotrypsin-β-Protein Complex and Synthetic Peptides for UseTherein," by Huntington Potter (Abandoned). Priority is also claimed toPCT/US93/00325 filed Jan. 13, 1993. The teachings of of theseapplications are incorporated herein by reference.

BACKGROUND

Alzheimer's disease is a degenerative disorder of the central nervoussystem that results in a progressive loss of memory and otherintellectual functions, such as reasoning, orientation, and judgement(Katzman, R., "Biological Aspects of Alzheimer's Disease," BanburyReport 15, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,(1983)). Alzheimer's disease occurs in sporadic and familial forms and,in the United States, affects about 600 people for every 100,000.

The earliest stages of Alzheimer's disease are characterized by"pre-amyloid" deposits of Alzheimer's β-peptide (Aβ), which areamorphous deposits of Aβ found in many regions of the brain. Aβ is a39-42 amino acid peptide that is derived from an about 700 amino acidcellular protein of unknown function (Glenner, G. G and Wong, C. G.,Biochem. Biophys. Res. Commun., 120:885-890 (1984)). As the diseaseprogresses, neuritic plaques form in the higher centers of the brain,particularly the hippocampus, frontal cortex and amygdala, and alsoaccumulate in the walls of cerebral and meningeal blood vessels moregenerally. These neuritic plaques consist of mature amyloid depositswhich, when viewed in the electron microscope, appear as large numbersof 6-10 nm diameter filaments consisting of Aβ. Amyloid deposits exhibitcertain characteristic staining properties (Abraham, C. R. et al., Cell,52:487-501 (1988)).

In addition to Aβ, mature Alzheimer amyloid deposits contain otherproteins, in particular the protease inhibitor α₁ -antichymotrypsin (α₁-ACT) (Abraham, C. R. et al., Cell, 52:487-501 (1988) and the lipidcarrier protein Apolipoprotein E (ApoE) (Namba, Y. et al. BrainResearch, 541:163 (1991) and Frangione, B., Neurosci. Lett. 135:235(1992)). Surrounding the mature amyloid deposits is a halo ofdegenerating neurites.

Aβ is apparently derived from a larger membrane-spanning precursorprotein whose RNA is alternately spliced to yield several proteinproducts (Seikoe, D. J., Science, 248:1058-1060). These observationssuggested that the amyloid deposits in Alzheimer's disease could resultfrom abnormal expression or posttranslational modification or processingof a normal molecule. Also intriguing was the finding that the geneencoding the amyloid protein precursor is located on chromosome 21,suggesting a common cause for the deposits observed in Down syndrome,caused by trisomy of chromosome 21, and Alzheimer's disease.

As mentioned above, some cases of Alzheimer's disease appear to befamilial, and are inherited in an autosomal dominant fashion. Linkageanalysis in four families pointed to a lesion on the long arm ofchromosome 21 (St. GeorgeHyslop, P. H. et al., Science, 238:664-660(1987)), which correlated well with the mapping data and similaritiesbetween Down syndrome and Alzheimer disease. Recently, hereditarycerebral hemorrhage with amyloidosis of Dutch origin was reported to belinked to the APP gene, and a point mutation in the coding region of thegene was identified (Van Broeckhoven, C. et al., Science, 248:1120-1122(1990); Levy, E. et al., Science, 248:1124-1126 (1990)). Patients withthis disease have a form of the β-protein in amyloid deposits inmeningeal and cerebral blood vessels.

However, other studies reported linkage of familial Alzheimer's diseaseto a locus on chromosome 21 distinct from the amyloid precursor protein(APP) gene (Tanzi, R. E. et al., Nature, 329:156-157 (1987); VanBroeckhoven, C. et al., Nature, 329:153-155 (1987)). Furthermore, therewas no evidence of duplication of the APP gene in cases of familial orsporadic disease. In fact, studies of some families reportedly indicateno linkage to chromosome 21 (Schellenberg, G. D., Science, 241:1507-1510(1988)). These data suggest that there may be genetic heterogeneity inthe cause of inherited forms of Alzheimer's disease, and other locationsfor the disease gene have been proposed, such as chromosome 14(Weitkamp, L. R., Amer. J. Hum. Genet., 35:443-453 (1983) andSchellenberg, G. D. et al. Science, 258:668 (1992)).

Thus, other components of neuritic plaques that are associated with themature amyloid deposits may also be of interest and may provide clues tothe cause or progress of the disease. These components may also beinvolved in the neuropathology of the disease and consequently mayprovide targets for therapeutic drugs which slow the progress oralleviate the symptoms of the disease.

SUMMARY OF THE INVENTION

The present invention relates to methods of screening for drugs whichcan be used to treat Alzheimer's disease. It further relates to methodsof treating an individual with Alzheimer's disease. The presentinvention also relates to methods of suppressing the formation ofneurotoxic Aβ filaments which are present in brain cells of individualswith Alzheimer's Disease and methods of slowing the progression of thedisease. It further relates to peptides which suppress the formation ofneurotoxic Aβ filaments. The present invention is based, in part, on thediscovery that α₁ -ACT, Apolipoprotein E4 (ApoE4), Apolipoprotein E3(ApoE3) and Apolipoprotein E2 (ApoE2) promote the assembly of Aβ intoamyloid filaments, and that α₁ -ACT and ApoE4 promote the assembly of Aβinto amyloid filaments which result in neuronal cell death. ApoE4 is themost efficient promoter of the formation of amyloid filament. ApoE2 isthe least efficient promoter and even inhibits the formation of amyloidfilaments in the presence of Aβ and ApoE4. This invention is also basedon the discovery that astrocytes from areas of the brain which aresusceptible to developing mature amyloid plaques over-express α₁ -ACT inthe presence of the higher than normal levels of interleukin 1 (IL-1)which are present in the brains of individuals with Alzheimer's disease.

One embodiment of the method of screening is an assay for identifyingcompounds which slow the formation of Aβ filaments from Aβ and α₁ -ACT,Aβ and ApoE3 or Aβ and ApoE4. Another embodiment of the method ofscreening is an assay for identifying compounds which suppress neuronalcell death in culture in the presence of Aβ and α₁ -ACT or Aβ and ApoE4.A third embodiment of the method of screening is an assay foridentifying compounds which suppress the release of α₁ -ACT from aculture of astrocytes in the presence of IL-1.

In one embodiment of the method of treating an individual withAlzheimer's disease, a therapeutically effective dose of a compoundwhich interferes with the Aβ/α₁ -ACT or Aβ/ApoE4 interaction isadministered to the individual. In another embodiment of the method oftreating an individual with Alzheimer's disease, a therapeuticallyeffective dose of a compound which suppresses neuronal cell death byinterfering with the Aβ/α₁ -ACT or Aβ/ApoE4 interaction is administeredto the individual. In yet another embodiment of the method of treatingan individual with Alzheimer's disease, a therapeutically effective doseof a compound which interferes with the ability of astrocytes from areasof the brain prone to develop mature amyloid plaques to over-express α₁-ACT is administered to the patient.

Another embodiment of the present invention refers to a compositioncomprising Aβ and a promoting factor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphic representation of the rate of in vitro formation ofAβ filaments over time from Aβ, as monitored by light scattering.

FIG. 2 is a graphic representation of the rate of the formation in vitroof Aβ filaments over time from a mixture Aβ and α₁ -ACT compared withthe rate of formation of the filaments from Aβ alone and from a mixtureof Aβ and the protein BSA. The rate of formation is monitored by lightscattering.

FIG. 3 compares in tabular form the amount of Aβ filaments formed invitro from the mixture of Aβ and α₁ -ACT, ApoE2, ApoE3, ApoE4Apolipoprotein AI (ApoAI) or Apolipoprotein AII (ApoAII). The formationof the filaments was determined by quantitative electron microscopy.

FIG. 4 compares in tabular form the neurotoxicity of Aβ filaments tohuman cortical neurons formed from the reaction of Aβ and variouspromoting factors.

FIG. 5 shows in tabular form the ability of Aβ₁₋₁₂ to suppress theneurotoxicity of the combination of Aβ and α₁ -ACT towards neuron-likePC-12 cells.

FIG. 6 shows in tabular form the ability of Aβ₁₂₋₂₈ to suppress theneurotoxicity of the combination of Aβ and ApoE4 towards neuron-likePC-12 cells.

FIG. 7 shows that the pathological chaperones, ACT and apoE4, promotethe toxicity of Aβ1-42 in human cortical neurons in culture.

FIGS. 8a and 8b show the neuroprotection effect of anti-pathologicalchaperons, Aβ-related peptides and apoE2, on human cortical neurons inculture from pathological effects of ApoE4 and ACT respectively.

FIG. 9a is a graph showing the effect of the Aβ₁₂₋₂₈ concentrations onapoE4 neurotoxic Aβ1-42 filaments-promoting activity.

FIG. 9b is a graph showing the effect of the Aβ₂₋₉ concentrations on ACTneurotoxic Aβ1-42 filaments-promoting activity.

FIG. 9c is a graph showing the effect of the ApoE2 concentrations onApoE4 neurotoxic Aβ1-42 filaments-promoting activity.

DETAILED DESCRIPTION OF THE INVENTION

As described herein, α₁ -ACT and Apolipoprotein E are not merelyincidently associated with mature amyloid plaque that is indicative ofAlzheimer's disease. They also promote the formation of Aβ filaments andplay a role in causing these filaments to become neurotoxic.

As described in Example 1, Aβ 1-42 can spontaneously assemble into highmolecular weight amyloid-like filaments in vitro. The formation of thesefilaments can be monitored by light scattering and electron microscopy,which show that filaments of about 6 nm wide and up to about a fewhundred nm long steadily increase in number.

Although Aβ can form amyloid-like filaments spontaneously, suchfilaments are formed much more rapidly in the presence of the amyloidassociated protein α₁ -ACT, as described in Example 2. In the presenceof α₁ -ACT, filaments form in a matter of hours, rather than days, andcan grow to very great lengths, frequently longer than 1 μM andtraversing an entire electron microscope grid square. The diameter ofthese filaments appears to be identical to those formed by Aβ alone orfound in tissue sections from Alzheimer's disease brain. Quantitation bylight scattering (which measures the total mass of material in filamentform) and by counting random filament crossovers in the electronmicroscope (a measure of both filament number and length) indicates thatα₁ -ACT accelerates the rate of filament formation at least tenfold. Assoon as eight hours after mixing Aβ and α₁ -ACT, filament formationappears to reach a maximum under the conditions used in Example 1.

α₁ -ACT is a serine protease inhibitor. The N-terminal 12 amino acids ofAβ resemble the active site of serine proteases. These structuralelements, coupled with enhancement of the rate of Aβ filament formationin the presence of α₁ -ACT, suggest the formation of a complex betweenα₁ -ACT and Aβ. The formation of this complex was demonstrated in thedisclosure of U.S. Ser. No. 08/179,574, the contents of which have beenexpressly incorporated into this application in their entirety.Chymotrypsin is a serine protease which can be inhibited by α₁ -ACT.U.S. Ser. No. 08/179,574 discloses that a peptide corresponding to aminoacids 1-12 or 1-28 of the N-terminal of Aβ peptide (hereinafter referredto as "Aβ₁₋₁₂ " and "Aβ₁₋₂₈ ", respectively) suppress the ability of α₁-ACT to inhibit chymotrypsin, while a control peptide from amino acids258-277 of the Aβ had no effect on α₁ -ACT activity. Aβ₁₋₁₂ and Aβ₁₋₂₈form stable complexes with α₁ -Act, presumably explaining the inhibitoryproperties of these peptides. Apparently the N-terminal amino acidresidues of Aβ bind with the active site of α₁ -ACT as apseudosubstrate, rendering it inaccessible to its normal substrates,such as chymotrypsin. The binding between these two proteins isspecific, as evidenced by the ability of the complex to survive harshtreatments, such as boiling in SDS and mercaptoethanol.

ApoE, like α₁ -ACT, is also present in Alzheimer amyloid deposits andbinds tightly to the Aβ protein in vitro. Recent epidemiological studieshave indicated that the development of Alzheimer's disease in severalfamilies with the "late-onset," inherited form of the disorder dependsin part on the particular ApoE alleles carried by the individual. Onsetof the disease occurs at a much earlier age in individuals with one ortwo copies of the ApoE4 allele than in those with the more commonApolipoprotein E3 (ApoE3) allele. Also, genotypes containing the E2allele are observed less frequently in Alzheimer Disease patients thanin control subjects (Corder, et al., Nature Genetics, 7:180 (1994)).Consequently, it is of interest to determine precisely what role ApoE4,ApoE3 and ApoE2 have in the etiology of Alzheimer's disease.Accordingly, the ability of purified ApoE4, ApoE3 and ApoE2 to promoteAβ filament formation in vitro was determined. As shown in Examples 3and 4, ApoE2, ApoE3 and ApoE4 promote Aβ filament formation; the ApoE4isoform was more active than the ApoE3 or ApoE2 isoform. Electronmicroscopic examination of the reaction of Aβ with ApoE4 showed thepresence of large numbers of long amyloid-like filamentsindistinguishable in appearance from those generated in the presence ofα₁ -ACT. Fewer filaments were observed if ApoE3 and ApoE2 were insteadadded to the reaction. Quantitation of the electron microscopicphotographs of filaments confirmed that ApoE4 is most effective inpromoting Aβ filament assembly, with α₁ -ACT and ApoE3 exhibitingintermediate catalytic activity. ApoE2 was the least effective inpromoting Aβ filament assembly of the apolipoproteins assessed. Twoother Apolipoproteins, ApoAI and ApoAII, were inactive as promotingfactors. The conclusions of these experiments are that the twoamyloid-associated proteins, α₁ -ACT and ApoE, particularly the ApoE4isoform, promote the formation of Aβ filaments from the Alzheimer Aβpeptide in vitro.

The presence of mature amyloid plaques in the brains of Alzheimerpatients and the absence of these plaques in disease free individualssuggests that their presence may in some way be responsible for theneuronal cell death that characterize this disorder. Certain aspects ofthe present invention are based on the discovery that certain componentsof mature amyloid plaque, i.e. α₁ -ACT and ApoE4, are responsible forthe formation of Aβ filaments that results in neural cell death. Celldeath may result from Aβ filaments that are themselves neurotoxic orfrom the process by which the Aβ filaments form. This discovery was madeby investigating the potential activity of Aβ filaments formed in thepresence of α₁ -ACT, ApoE3 and ApoE4 on human cortical neurons andneuron-like PC-12 cells in culture. As used herein "neuron-like PC-12cells" are PC-12 cells that have stopped dividing and havedifferentiated into neuron-like cells as a result of the addition ofnerve growth factor (NGF).

Reaction mixtures containing Aβ show minimal neurotoxicity againstprimary human cortical neurons. However, as described in Example 4, thereaction mixtures containing Aβ and α₁ -ACT or Aβ and ApoE4 were highlyneurotoxic to human cortical neurons after preincubation, whereas thereaction mixtures containing Aβ and ApoE3 were not. Similar increases inneurotoxicity towards neuron-like PC-12 cells were observed withreaction mixtures containing Aβ and ApoE4 or Aβ and α₁ -ACT. Althoughthe treated cultures showed only minor morphological changes, such as anincrease in rounded cells that had become detached from the substrate, abiochemical assay that uses the metabolizable, chromogenic substrate 3,(4,4-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT) toquantitatively measure viable cells indicates a strong neurotoxic effectof the filaments formed in vitro. Hansen M. B. et al., J. Immunol.Methods, 119:203 (1989). The ApoE3 isoform, which is associated with amuch later age of onset of familial Alzheimer's disease, promotes theformation of apparently similar Aβ filaments, but they are notneurotoxic to human cortical neurons. Furthermore, ApoE3, when includedin the reaction mixture with Aβ and ApoE4, results in Aβ filaments withconsiderably reduced neurotoxicity. Separation of the filaments formedin the presence of α₁ -ACT or ApoE4 from unpolymerized proteins bycentrifugation indicated that the filaments (or possibly short,sedimentable oligomers) are the toxic agent. In sum, these resultsindicate that the amyloid-associated proteins α₁ -ACT, ApoE4, ApoE3 andApoE2 promote the formation of neurotoxic Aβ filaments and, thus, canfunction as pathological chaperones or "promoting factors." As definedherein, "promoting factors" are amyloid associated proteins that, in thepresence of Aβ, increase the rate of formation of Aβ filaments. Asdiscussed, neurotoxicity results when Aβ filaments are formed in thepresence of certain promoting factors such as α₁ -ACT and ApoE4.

The presence of proteins other than Aβ in the mature amyloid plaques ofAlzheimer's disease has often been thought to represent adventitiousbinding to already formed Aβ filaments. This hypothesis was reinforcedby the finding that synthetic Aβ peptide could, under certainconditions, form filaments spontaneously in vitro. However, the resultspresented herein clearly show that this hypothesis is incorrect. Theability of α₁ -ACT and ApoE4 to hasten the formation of Aβ filaments andto cause these filaments to become neurotoxic shows that these proteinsdo more than merely adventitiously bind to amyloid plaque. Rather, theyact as promoting factors to further the progress of the disease and tocause, at least in part, its neurodegenerative symptoms. The molecularmechanism by which α₁ -ACT and ApoE4 promote neurotoxic Aβ filamentformation has yet to be determined, but must involve direct interactionbetween the participating proteins.

The role which α₁ -ACT and ApoE4 play as promoting factors inAlzheimer's disease suggests that drugs which block the interaction ofthese proteins with Aβ can slow the progress of the disease and suppressneurodegeneration. Such drugs can be identified through an assay whichmeasures the ability of a compound to disrupt the interaction amongApoE4, ApoE3, ApoE2, α₁ -ACT and Aβ. Support for this approach withrespect to the Aβ/α₁ -ACT interaction has been provided by results,presented herein, which show that peptides comprised of Aβ₁₋₁₂ or Aβ₁₋₂₈block the ability of α₁ -ACT to inhibit chymotrypsin. These results showthat synthetic analogs of Aβ can divert α₁ -ACT from its harmfulinteractions. With respect to the interaction between Aβ and ApoE4, theresults which show that the formation of Aβ filaments is inhibited whenApoE2 and ApoE4 are incubated with Aβ (FIG. 3 and Example 3) arerelevant. Apparently, ApoE2 is able to disrupt the interaction betweenApoE4 and Aβ. This result is consistent with the observation thatgenotypes containing the E2 allele occur less frequently in Alzheimerpatients than in controls (Corder, et. al., Nature Genetics, 7:180(1994)). In addition, Aβ filaments assembled from Aβ, ApoE4 and ApoE3are less neurotoxic than Aβ filaments assembled from only Aβ and ApoE4are relevant. Apparently, ApoE3 is able to interact with Aβ, therebyblocking the more neurotoxic Aβ/ApoE4 interaction. Furthermore, Aβfilaments promoted by ApoE3 are not neurotoxic, but those promoted byApoE4 are neurotoxic.

Further support for this method was provided by investigating whetherthe neurotoxic effects of mixtures of Aβ and proteins which arepromoting factors can be blocked by peptides which bind the promotingfactors. For example, ApoE4 is thought to bind a peptide comprisingamino acid residues 12-28 of Aβ (hereinafter referred to as "Aβ₁₂₋₂₈ "),a region of the peptide predicted to be prone to forming a β-pleatedsheet (Strittmatter, et al., Proc. Natl. Acad. Sci. USA, 90:8098(1993)). A mixture of ApoE4, Aβ and Aβ₁₂₋₂₈ showed approximately afour-fold reduction in neurotoxicity towards neuron-like PC-12 cells inculture compared with the same cultures of neuron-like PC-12 cellsincubated in the absence Aβ₁₂₋₂₈ (see Example 5 and FIG. 5). Similarresults were observed when neuron-like PC-12 cells were incubated withAβ, α₁ -ACT and Aβ₁₋₁₂ and compared with the identical PC-12 culturesincubated in the absence of Aβ₁₋₁₂ (see Example 5 and FIG. 6). Theseresults provide support for the use of compounds that interfere with theAβ/ApoE4 or Aβ/α₁ -ACT interaction as effective therapeutics which canslow neurodegeneration and the formation of Aβ filaments in anindividual with Alzheimer's disease. They also provide the basis for anassay which assesses the ability of compounds to interfere with theinteraction between Aβ and these promoting factors and, thus, providesan effective means of identifying such therapeutics.

It has also been discovered that protection against neurotoxocity can beprovided by peptides shorter than Aβ₁₋₁₂ and Aβ₁₂₋₂₈. For example, a 2.0to 2.4-fold reduction in neurotoxicity is observed when human corticolneurons cells incubated with a mixture of α-ACT, Aβ and Aβ₂₋₉ than whenthe cells are incubated with a mixture Aβ and α₁ -ACT alone (Example12). As described herein, amino acid sequences within Aβ₁₋₁₂ and Aβ₁₂₋₂₈(shorter than Aβ₁₂₋₂₈) are useful for inhibiting respectively, theneurotoxic effects of α₁ -ACT together with Aβ and Apolipoprotein E4together with Aβ. In one embodiment, amino acid residues present inAβ₂₋₉ which can be amino acid residues 2-9 or fewer amino acid residues)are useful as inhibitors. However, it is clear that oligopeptides longerthan eight amino acids, and indeed longer than twelve amino residueswhich contain the critical residues can bind α₁ -ACT and inhibitneurotoxicity. Similarly, oligopeptides both longer and shorter thanAβ₁₂₋₂₈ can, if they contain the critical amino acid residues, bindApolipoprotein E4 and thereby inhibit neurotoxicity.

α₁ -ACT and ApoE4 are expressed in various parts of the body. Forexample, the fact that Aβ-containing amyloid deposits that form in thewalls of cerebral blood vessels in Alzheimer's disease, Down syndromeand in a cerebral hemorrhage disease (HCHWAD) are all of the maturefilamentous form may reflect the ready availability of α₁ -ACT and ApoE4in the circulation. However, if α₁ -ACT and ApoE4 are to act aspromoting factors for Alzheimer's disease, they must be expressed insufficiently high quantities within the brain that they come in contactwith Aβ. Within the brains of normal individuals, α₁ -ACT and ApoE areproduced by astrocytes, as evidenced by immunocytochemical analysis(Abraham et al., Neurobiol. Aging, 11:123 (1990) and Koo et al.,Neurobiol. Aging, 12:495 (1991)). However, in situ hybridization,Northern blots, and Western blots have shown that α₁ -ACT wRNA andprotein in astrocytes are greatly increased in those areas of Alzheimerbrain prone to amyloid deposition, particularly the hippocampus, frontalcortex and amygdala (Pasternack et al., Am. J. Path., 135:827 (1989),Abraham et al., Neurobiol. Aging, 11:123 (1990), Koo et al., Neurobiol.Aging, 12:495 (1991) and Rebeck et al., Neuron, 11:575 (1993)). Theseresults are consistent with α₁ -ACT and ApoE4 acting as factors thatpromote the deposition of mature amyloid plaques, but they do notexplain what causes the over-expression of α₁ -ACT and ApoE4.

Certain aspects of the present invention are based on the discovery thatthe cytokine interleukin 1 (IL-1), which is over-expressed inAlzheimer's disease, functions to induce α₁ -ACT expression in humanbrain astrocytes in culture. Mixed cultures of astrocytes and microglialcells prepared from human fetal brain express α₁ -ACT in response toexogenously added IL-1 (see Example 6). When the mixed cultures ofastrocytes and microglial cells are prepared from areas of human brainthat are prone to developing mature amyloid pathology, such as thefrontal cortex or the hippocampus, the astocytes spontaneously expressα₁ -ACT as the cultured cells reach confluence. Confluence refers tocells in culture which have grown to the extent that there is noremaining space left between the cells. Confirmation that astrocytes areindeed the cells which produce α₁ -ACT in response to IL-1 is providedby the observation that the same level of α₁ -ACT production is inducedby a given concentration of exogenously added IL-1 when microglial cellsare removed from the mixed cultures as in mixed cultures retaining themicroglial cells.

The release of α₁ -ACT from astrocytes in confluent mixed cultures isapparently in response to IL-1 released from the microglial cells.Support for this proposal comes from the observation, discussed above,that the same amount of α₁ -ACT mRNA is produced in mixed cultures ofastrocytes and microglial cells compared with pure astrocytes inresponse to the same amount of IL-1 (see Example 10). Because there areno significant differences in the number of microglia removed fromcortical and cerebellar cultures (see Example 10), it seems likely thatthe difference between these two cultures resides in the ability ofmicroglial cells from the cortical region to secrete and/or synthesizeIL-1. This proposal is consistent with the observation that there arefewer IL-1 positive microglial cells in normal cortex than inAlzheimer's brain cortex (Griffin, et al., Proc. Natl. Acad. Sci. USA86:7611 (1989)). Support for this proposal is also provided by theobservation that mixed cultures of astrocytes and microglial cells fromnon-susceptible areas of the brain, such as the cerebellum or the brainstem, must be induced to express α₁ -ACT by addition of exogenous IL-1(see Example 8). In addition, fewer IL-1 positive cells are found in thecerebellum than the cortex of Alzheimer's brain (see Example 11).

This regional specificity of IL-1 release, the differential responses ofthese regions to IL-1, along with the other results presented herein,support the conclusion that the over-expression and release of α₁ -ACTand ApoE4 are factors in accelerating the formation of mature amyloidfilament deposits.

The release of α₁ -ACT from astrocytes originating from susceptibleareas of the brain suggests that the deposition of mature amyloidplaques and the formation of neurotoxic Aβ filaments can be suppressedby interfering with the response of these astrocytes to IL-1. Supportfor the proposal is provided by the discovery that antibodies whichblock the IL-1 receptor prevent the spontaneous production of α₁ -ACT inconfluent mixed cultures of astrocytes and microglial cells preparedfrom areas of the human brain that are prone to developing matureamyloid pathology (see Example 9). These results provide the basis foran effective method of screening for prospective drugs to treatAlzheimer's disease; such an assay determines whether contactingastrocytes with IL-1 and in the presence and in the absence (control) ofa compound being screened results in less release of α₁ -ACT from theastrocytes in the absence of the compound than in its presence.Alternatively, the assay determines whether confluent astrocytes andmicroglial cells which have been isolated from areas of the brain thatare susceptible to developing mature amyloid plaques release less α₁-ACT in the presence of a compound being screened than in the absence ofthe compound. The results discussed above also suggest that theformation of neurotoxic Aβ filaments and deposition of nature amyloidplagues can be suppressed by interfering with the ability of microgliain susceptible areas of the brain, e.g. the cortex, to release and/orproduce IL-1. The assay can also determine whether contacting the cellmixture with the compound being screened results in a decrease in IL-1release from the microglial cells.

The release of interleukin 1 from microglial cells in the brain shouldlead to altered levels in interleukin in biological fluids outside ofthe brain, e.g. the cerebrospinal fluid and blood. Determining alteredlevels of interleukin 1 in these biological fluids thus provide a methodof detecting Alzheimer's disease in an individual. It is also a methodof diagnosing Alzheimer's disease before the onset of symptoms normallyassociated with the disease.

The present invention relates to methods of identifying compounds whichinhibit the processes involved in the progression of Alzheimer'sdisease. These processes include the formation of Aβ filaments, theenhanced ability of astrocytes from susceptible areas of the brain torelease α₁ -ACT in response to IL-1 and neuronal cell death resultingfrom the formation of Aβ filaments. The present invention furtherrelates to methods of inhibiting these processes.

One embodiment of the present invention is a method of screening forcompounds which inhibit the formation of Aβ filaments. "Compound" in thepresent invention refers to small organic or inorganic molecules,oligopeptides or peptides, as well as to molecules designed to mimic thestructure or function of peptides or oligopeptides. For example,specific RNA molecules can be developed and screened for activityagainst enzymes. The method comprises contacting the compound beingscreened for its ability to inhibit the formation of Aβ filaments withAβ and one or more amyloid associated proteins which promote theformation of Aβ filaments, under conditions suitable for the formationof Aβ filaments. This mixture is referred to as the test sample. Amyloidassociated proteins which promote the formation of Aβ filaments includeα₁ -ACT, ApoE2, ApoE3 and ApoE4. The method further comprisesdetermining the amount of Aβ filaments formed in the test sample andcomparing the amount formed to a suitable control. The control sample isrun identically to the test sample, except that the control is run inthe absence of the compound being tested for its ability to inhibit theformation of Aβ filaments. The control can be run simultaneously withthe test sample. Alternatively, the control is run prior to or after thetest sample, in which case it is used as pre-determined standard. Theformation of a lesser amount of Aβ filaments in the presence of thecompound being screened compared with the amount formed in the controlindicates that the compound being screened inhibits the formation of Aβfilaments. Determining the extent to which Aβ filaments form in the testsample and in the control sample can be carried out by methods known tothose skilled in the art, including electron microscopy. Electronmicroscopy can be used to quantitatively determine the amount of Aβfilament formed. Alternatively, electron microscopy can be used toapproximate the amount of Aβ filaments formed, for example by visualapproximation, or to determine the presence or absence of Aβ filamentsin the test sample. Light scattering can also be used to determine thedegree of filament formation. Differential precipitation or filtrationof the reaction in which Aβ is used having a radioactive label,fluorescent label, a chromogenic label or an enzyme label that can bedetected by its activity are also techniques which can be employed tomeasure Aβ filament formation.

Another embodiment of the present invention is a method of identifyingan inhibitor of the formation of Aβ filaments or a compound whichsuppresses neurotoxic filament formation by identifying a compound whichbinds α₁ -Act (or ApoE4) such that α₁ -Act (or ApoE4) no longer bindsAβ. An inhibitor of the formation of Aβ filament formation or asuppressor of Aβ filament neurotoxicity can be identified by combiningα₁ -ACT (e.g. in solution or bound to solid support), chymotrypsin, achymotrypsin substrate (i.e. a peptide which can be cleaved bychymotrypsin) and the compound being assessed to give a testcombination. The test combination is subjected to conditions suitablefor cleavage of the substrate. The amount of cleaved substrate is thenassessed by methods known in the art (e.g. by use of a chromogenicsubstrate, a radiolabeled substrate or a biotinylated substrate) andcompared to a suitable control. A suitable control is the testcombination without the compound being assessed, subjected to theconditions suitable for cleavage of the substrate. If the cleavage ofthe chymotrypsin substrate occurs to a greater extent in the testcombination than in the control, the compound being assessed binds α₁-ACT, thereby inhibiting the formation of Aβ filaments and suppressingthe neurotoxicity of Aβ filaments.

Another embodiment of the present invention is a method of screening fora compound which suppresses neuronal cell death in Alzheimer's patients.This method comprises contacting a first culture of neuron orneuron-like cells with Aβ, one or more amyloid associated proteins whichpromote the formation of neurotoxic Aβ filaments, and a compound beingscreened for its ability to suppress neuronal cell death in anAlzheimers patient. The first culture is contacted with these componentsunder conditions suitable for neuronal cell survival. The degree of celldeath is assessed and compared to a suitable control. A suitable controlis a second culture of the neuron-like cells, Aβ and the one or moreamyloid associated proteins which promote the formation of neurotoxic Aβfilaments. The second culture of neuron-like cells is contacted withthese components under conditions suitable for cell survival. Theseconditions are identical with the conditions used in the screening beingperformed with the first culture, except that the compound beingscreened for its ability to inhibit neuronal cell death is not presentin the control. The control can be run simultaneously with the screeningbeing performed with the first culture. Alternatively, the control isrun prior to or after the screening being performed with the firstculture, in which case it is a pre-determined standard. Less cell deathin the first culture as compared with cell death in the control isindicative of a compound which can suppress neuronal cell death in thebrain of an Alzheimer patient. The degree of cell death or cell toxicitycan be determined by a number of methods known to those skilled in theart, including assaying apotosis by nucleic acid degradation usingstandard commercially available kits, an MTT release assay, visualinspection in the light microscope and Trypan blue exclusion. The degreeof cell death can be determined quantitatively, approximated ordetermined by the presence or absence of neuronal cell death.

Neuron cells or neuron-like cells may be used in the method of screeningfor a compound which suppresses neuronal cell death in Alzheimer'spatients include neurons from human or animal brains. Preferred neuronalcells are human cortical neurons. As defined herein, neuron-like cellsare those cells which have one or more characteristics of neuron cells,including action potentials, processes and the ability to releaseneurotransmitters. Preferred neuron-like cells are those that haveprocesses. Suitable neuron-like cells include continuously growing celllines that have been differentiated into neuron-like cells with nervegrowth factor (NGF) or a differentiation factor such as retinoic acidcan be used. Suitable continuously growing cell lines includeneuroblastoma cells, cells formed by a fusion of neuroblastoma cells andneurons and P19 embryo carcinoma cells. A preferred continuous growingcell line is the PC-12 cell line.

A further embodiment of the present invention is a method of screeningfor compounds which suppress the formation of neurotoxic Aβ filaments.This method comprises contacting a first culture of astrocytes cellswith IL-1 and a compound being screened for its ability to suppress theformation of neurotoxic Aβ filaments. Alternatively, the first culturecan be a mixed culture which includes astrocytes and microglial cellswhich have been isolated from areas of the brain which are prone todeveloping mature amyloid pathology and which have reached confluence.Areas of the brain prone to developing mature amyloid pathology includethe hippocampus, the frontal cortex and the amygdala. Reachingconfluence refers to astrocytes and microglial cells in culture whichhave grown to the point where there is no further space for growth leftbetween the cells. Because microglial cells in these confluent mixedcultures spontaneously produce IL-l, there is no need to add exogenousIL-1 if the mixed culture is used in the assay. The amount of α₁ -ACTproduced in the first culture is determined and compared to the amountα₁ -ACT produced in a second culture of astrocytes and microglial cellsthat serves as a control culture. Alternatively, the amount of IL-1produced in the first culture by the microglial cells in determined andcompared to the amount of IL-1 produced by the microglial cells in thesecond culture. The microglial cells may also be replaced with otherIL-1 secreting cells. The control is performed identically to the firstculture, except that the culture is not contacted with the compound ormolecule being screened for its ability to inhibit the formation ofneurotoxic Aβ filaments. The production of less α₁ -ACT or less IL-1 inthe first culture compared with the second culture is indicative thatthe compound being screened suppresses the amount of neurotoxic Aβfilament formation in the brains of Alzheimer's patients. The amount ofα₁ -ACT or IL-1 produced in the control can be assessed simultaneouslywith the screening of a compound in the first test culture.Alternatively, the control can be run prior to or after screening beingperformed with the first test culture, and they serve as a predeterminedcontrol.

The amount of α₁ -ACT produced can be assessed by methods known to thoseskilled in the art, for example by a Northern blot analysis of the totalRNA produced by the astrocytes using autoradiography with radioactive α₁-ACT cDNA as a probe. The α₁ -ACT RNA hybridizes with the α₁ -ACT cDNA,which allows the amount of α₁ -ACT RNA present to be determined byexposing the blot to photographic film and then determining theintensity of the spot left on the film by the radioactive probe. Theamount of α₁ -ACT mRNA corresponds to the amount of α₁ -ACT that isbeing expressed by the astrocytes. The intensity of the spot can bedetermined quantitatively by densitometer analysis or phosphorimageranalysis of the original Northern blot, approximated, or determined bythe presence or absence of the spot. Alternatively, methods such as "dotblots" on nitrocellulose or nylon membranes or the like or ELISA assaysusing antibodies against α₁ -ACT protein can be used. The binding of alabeled substrate for α₁ -ACT such as chymotrypsin or a small peptidewhich should bind α₁ -ACT, such as Aβ or other peptides resembling theactive site of serine proteases, can also be used in ELISA-type assaysfor measuring the amount of α₁ -ACT protein generated by the astrocytes.

The amount of IL-1 produced can be analyzed by methods known in the art,including an assay employing a D10 cell line. These are cells which onlygrow in the presence of IL-1. D10 cells in a test sample are contactedwith an aliquot from a culture being tested for the presence of IL-1.Radiolabeled thymidine is added. A control sample is run identically tothe test sample, except that an aliquot from the culture being tested isnot added. After a suitable period of time, the DNA from both samplesare isolated and the amount of radioactivity present is determined. Lessradioactivity in the test sample than in the control sample isindicative that less IL-1 is produced in the culture being tested.Another embodiment of the present invention is a method of suppressingthe formation of neurotoxic Aβ filaments. The method consists ofcontacting Aβ and one or more amyloid associated proteins which promotethe formation of Aβ filaments with a compound that inhibits theformation of a complex between Aβ and the one or more amyloid associatedproteins. Compounds which inhibit the formation of a complex between Aβand α₁ -ACT include peptides comprising Aβ₁₋₁₂ and Aβ₁₋₂₈. They alsoinclude oligopeptides with a sufficient number of amino acid residues1-12 of Aβ that the oligopeptide binds α₁ -antichymotrypsin. Theoligopeptide can include amino acid residues which, in the Aβ peptideare contiguous or are non-contiguous. It can include Aβ₁₋₁₁, Aβ₁₋₁₀,Aβ₁₋₉, A62 ₂₋₁₁, Aβ₂₋₁₀ and Aβ₂₋₉. The amino acid residues can benaturally occurring or modified, e.g. the oligopeptide can be synthetic.Such compounds further include those peptides which mimic the bindingsite of serine proteases. Peptides which mimic the binding site ofserine proteases will typically have an Asp-Ser-Gly tripeptide, aconserved portion of the serine protease binding site, or its equivalentas part of the amino acid sequence. Potter et al., Ann. of the N.Y.Acad. Sci. 674:161 (1992). Compounds which inhibit the formation ofcomplexes between Aβ and α₁ -ACT also include synthetic peptides orcompounds sufficiently homologous to the binding site of α₁ -ACT thatthey bind Aβ. Compounds which inhibit the formation of a complex betweenAβ and ApoE4 include peptides comprising Aβ₁₋₂₈ and Aβ₁₂₋₂₈ andoligopeptides comprising a sufficient number of amino acid residues 1-12or 12-28 of Aβ that the oligopeptide binds α₁ -antichymotrypsin orApolipoprotein E4, respectively. Also included are synthetic peptides orcompounds sufficiently homologous to ApoE2 that they disrupt theinteraction between Aβ and ApoE4.

A further embodiment of the present invention also includes Aβ₁₋₂₈ andAβ₁₂₋₂₈ and peptides sufficiently homologous to Aβ₁₋₂₈ and Aβ₁₂₋₂₈ thatthey inhibit the formation of a complex between ApoE4 and Aβ. Suchpeptides include oligopeptides comprising a sufficient number of aminoacid residues 12-28 of Aβ that the oligopeptide binds Apolipoprotein E4.The oligopeptide can include residues which, in the Aβ, are contiguousor non-contiguous. The amino acid residues can be naturally occuring ormodified, e.g. the oligopeptide can be synthetic. ApoE4 appears to bindwith Aβ₁₂₋₂₈, which form a β-pleated sheet (Strittmatter et al., Proc.Natl. Acad. Sci., USA 90:8098 (1993)). The structural requirements of aβ-pleated sheet are well known in the art.

Consequently, the skilled artisan is able to choose variations in theamino acid sequence which would not alter the ability of the peptide toform a β-pleated sheet and synthesize these peptides by known peptidesynthesis methods. Such peptides would be equally effective in bindingApoE4, and would therefore similarly reduce the Aβ filament formationand neuronal cell death by disrupting the interaction between Aβ andApoE4. Such peptides are the subject of this invention. Synthetichomologues of Aβ₁₂₋₂₈ which inhibit the formation of a complex betweenApoE4 and Aβ can be identified by the methods of screening of thepresent invention.

Yet another embodiment of the present invention refers to compounds andsynthetic peptides sufficiently homologous to ApoE2 that they disruptthe interaction between Aβ and ApoE4.

Yet another embodiment of the present invention is a peptide comprisingAβ₁₋₁₂ and peptides sufficiently homologous to Aβ₁₂ that they bind α₁-antichymotrypsin and inhibit the formation of a complex between α₁-antichymotrypsin and Aβ. Included are oligopeptides with a sufficientnumber of amino acid residues 1-12 of Aβ that the oligopeptide binds α₁-antichymotrypsin. The oligopeptide can include amino acid residueswhich, in the Aβ peptide are contiguous or are non-contiguous. It caninclude Aβ₁₋₁₁, Aβ₁₋₁₀, Aβ₁₋₉, Aβ₂₋₁₁, Aβ₂₋₁₀ and Aβ₂₋₉. The a acidresidues can be naturally occurring or modified, e.g. the oligopeptidecan be synthetic. Such compounds further include those peptides whichmimic the binding site of serine proteases, as described above.

The skilled artisan is also able to modify active peptides by changingthe amino acid sequence, derivatizing amino acids within the sequence orincluding a non-amino acid or non-peptide structural element within thepeptide so as optimize desirable properties of the peptide. Suchpeptides can be prepared by known peptide synthesis techniques.Derivatization can be accomplished by techniques standard in the art oforganic chemistry. Desirable properties which can be designed into thesesynthetic peptides include optimizing its activity (e.g., its ability tointerfere with Aβ/ApoE4 interaction), solubility, ability to reach thetarget site in the brain and ability to avoid degradation within thebody. Methods of optimizing these and other properties are well knownwithin the art. For example, increasing the ability of the peptide tocross the blood brain barrier and reach the target site can beaccomplished by increasing the lipophilicity of the peptide.Alternatively, the peptide can be modified to include a component thatis recognized by a receptor on the blood-brain barrier and allows thepeptide to be internalized (brought across the blood-brain barrier) anddeposited into the neuropil. Optimizing activity, e.g., the bindingbetween ApoE4 and the synthetic peptide can be accomplished, forexample, by synthesizing peptide analogues of Aβ₁₂₋₂₈ and performingcompetitive binding assays between the synthetic analogue and Aβ₁₂₋₂₈.Synthetic peptides with improved ability to inhibit the formation ofneurotoxic Aβ filaments can be identified, for example, by synthesizinganalogs of ApoE2 and comparing the ability of those analogs with theability of ApoE2 to prevent the formation of Aβ filaments. The processof optimizing activity can be aided by rational based drug design, forexample by determining an x-ray crystal structure of a complex betweenApoE4 (or ApoE2) and Aβ₁₂₋₂₈ and identifying the significantinteractions between the two molecules. Optimizing the ability of thepeptide to avoid degradation within the body can be done by metabolicstudies in animals, for example by injecting radioactive analogous ofthe peptide and following their metabolic fate.

Aβ₁₂₋₂₈, Aβ₁₋₁₂ and Aβ₁₋₂₈, synthetic homologues of Aβ₁₂₋₂₈, Aβ₁₋₁₂,Aβ₁₋₂₈ and ApoE2 and oligopeptides comprising a sufficient number ofamino acid residues 1-12 or 12-28 of Aβ that the oligopeptide binds α₁-antichymotrypsin or Apolipoprotein E4, respectively, can be used in themethods of the present invention of treating an individual withAlzheimer's disease of the present invention. Alternatively, thesecompounds can be used in the methods of screening of the presentinvention to find new and more active compounds for treating anindividual with Alzheimer's disease. For example, Aβ₁₂₋₂₈ can be used asa standard in the method of screening for a compound which suppressesneuronal cell death in an individual with Alzheimer's disease. Compoundswhich are more effective in preventing cell death in this assay thanAβ₁₂₋₂₈ are potentially more effective therapeutics than Aβ₁₂₋₂₈ and canbe subjected to further testing. These compounds can also be used tofind new and more active compounds for preventing the formation of Aβfilaments. For example, ApoE2 can be used as a standard in the method ofscreening for a compound which inhibits the formation of Aβ filaments.Compounds which are more effective in preventing Aβ filament formationin this assay than ApoE2 are potentially more effective therapeuticsthan ApoE2 and can be subjected to further testing.

Other compounds which inhibit the formation of Aβ filaments includecompounds which inhibit the formation of a complex among Aβ, α₁ -ACT andApoE4. Such compounds include those compounds which inhibit theformation of Aβ/α-ACT and Aβ/ApoE4 complexes.

Another embodiment of the present invention is a method of suppressingneuronal cell death in the brain of an individual with Alzheimer'sdisease. This method comprises administering to the individual atherapeutically effective dose of a compound which interferes with theinteraction between Aβ and a promoting factor such as ApoE4 or α₁ -ACT.Interfering with this interaction includes, for example, a compoundwhich binds at or near the site on ApoE4 (or α₁ -ACT) where Aβ binds,thereby blocking Aβ from binding with ApoE4 (or α₁ -ACT). It is alsopossible to interfere with this interaction through the use of acompound which binds to ApoE4 (or α₁ -ACT) and changes the conformationof ApoE4 (or α₁ -ACT), thereby making it more difficult for Aβ to bindto ApoE4 (or α₁ -ACT). Suitable compounds which bind ApoE4, therebypreventing ApoE4 and Aβ binding, include a synthetic peptide whichcomprises amino acid residues Aβ₁₋₂₈ or Aβ₁₂₋₂₈. Suitable compounds alsoinclude oligopeptides comprising a sufficient number of amino acidresidues 1-12 or 12-28 of Aβ that the oligopeptide binds α₁-antichymotrypsin or Apolipoprotein E4, respectively, as describedabove. Interfering with the interaction between ApoE4 (or α₁ -ACT) andAβ also includes a compound which binds at or near the site where ApoE4(or α₁ -ACT) binds to Aβ, or at a site which changes the conformation ofAβ, thereby inhibiting ApoE4 (or α₁ -ACT) from binding to Aβ.

Yet another embodiment of the present invention is a method ofsuppressing the formation of neurotoxic Aβ filaments. In thisembodiment, astrocytes and microglia cells from areas of the brainsusceptible to developing mature amyloid pathology are contacted with acompound which inhibits the expression of α₁ -ACT by astrocytes.Inhibiting the expression of α₁ -ACT by astrocytes, as defined herein,refers to inhibiting the transcription of the α₁ -ACT gene, preventingthe mRNA derived therefrom from being transported out of the nucleus orbeing translated, or preventing the transport of α₁ -ACT out of thecytoplasm of astrocytes.

Inhibiting the expression of the α₁ -ACT gene also refers to blockingthe biochemical pathway by which IL-1 communicates with the nucleus ofthe astrocytes, for example by blocking an IL-1 receptor on the cellsurface or reducing the levels of IL-1 in areas of the brain susceptibleto forming amyloid plaques. One way of interfering with the biochemicalpathway by which IL-1 communicates with the nucleus is by means ofmonoclonal antibodies which block the IL-1 receptor.

Monoclonal antibodies which block the IL-1 receptor are prepared byimmunizing a mouse or other suitable animal with IL-1 receptor protein.These proteins, preferably isolated from astrocytes which may showdifferent receptors than cells outside the nervous system, are used toimmunize mice by standard procedures. Following several boosterinjections of the immunogen and analysis of the ability of the recipientmouse to express antibodies to the IL-1 receptor, the animals aresacrificed, the spleens are harvested and disaggregated into individualcells, which include B lymphocytes. These cells are then fused to ahybridoma partner to immortalize them, and the fusion products plated inappropriate medium that will allow only the fused cells to grow intoindividual clones. Each clone will express a different antibody, and thecollection is screened for those clones expressing antibodies to theIL-1 receptor. Many of these antibodies will bind to the receptor buthave no effect on its function. A few will block the function of theIL-1 receptor, which can be assayed by their ability to prevent the D10cell line from responding to IL-1, or preferably screened by theirability to prevent purified human astrocytes from expressingantichymotrypsin in response to added recombinant IL-1. Cloned cellsexpressing the blocking antibodies to the IL-1 receptor are thenharvested, grown into large cultures, and used to prepare large amountsof the blocking antibody. This blocking antibody may be used for otherassays. The antibody may also be used as therapy for Alzheimer'sdisease. Specifically, the antibody should prevent the IL-1 directedacute phase response in the brain and the expression of ACT inastrocytes. Thus there will be less promotion of amyloid filamentformation and less neuronal cell death. Alternatively, the genes for theheavy chain and light chain components of the blocking antibody may beisolated by standard recombinant DNA technology from the mouse cellsexpressing the antibody. Portions of this gene that code for the IL-1binding area of the antibody may be subcloned and placed in the contextof the appropriate light or heavy chain human immunoglobin genes. Thecomposite genes can then be placed into an appropriate cell line forlarge scale expression of human blocking antibodies against the humanIL-1 receptor. These antibodies would be preferable therapeutic agentsinasmuch as they will contain only minimal, non-immunogenic regions ofthe original mouse antibody, and will therefore not be recognized andrejected by the human immune system.

Yet another embodiment of the present invention is a a method ofdetecting Alzheimer's disease in an individual. The method comprisesdetecting the level of interleukin 1 in the cerebrospinal fluid of theindividual, wherein an altered level of interleukin 1 in thecerebrospinal fluid of the individual is indicative of Alzheimer'sDisease in the individual. An "altered level of interleukin 1" meansthat amount of interleukin 1 found in a quantity of cerebrospinal fluidof the individual being assessed is quantitatively different (e.g.greater) than what is found in a suitable control, i.e. an individual orpopulation of individuals who do not have Alzheimer's disease.

Cerebrospinal fluid can be drawn from an individual by methods known inthe art. Interleukin 1 levels can be also be measured by methods knownin the art, for example by exposing the spinal fluid to radiolabeledantibodies specific for interleukin 1 and then exposing the resultingsolution to antibodies to a solid support which are specific for theradiolabeled antibodies. The amount of radioactivity bound to solidsupport is indicative of the amount of interleukin 1 in the sample.

Yet another embodiment of the present invention refers to a compositioncomprising Aβ and one or more promoting factors. Preferred promotingfactors include ApoE4 and α₁ -ACT. These compositions are prepared bymixing Aβ peptide and the promoting factor(s) under conditions suitablefor the formation of neurotoxic Aβ filaments. Suitable conditionsinclude molar ratios of promoting factor to Aβ, pH, ionic strengths andzinc ion concentrations which promote Aβ filament formation. Molarratios of Aβ to promoting factor can range from about 1:1 to about400:1; in one embodiment, it is 200:1 and in another is 4:1. pH canrange from about 6 to about 8; typically pH 7.0 is used. Ionic strengthcan range about 1 μM to about 0.150 M, but 10 μM is preferred. Zinc ionconcentration can range from 0 to about 100 μM, but 25 mM is preferred.Glycine may optionally be added at concentrations from about 0 to about1.0 mM. Specific conditions are described in Examples 1, 2 and 4. Thesenovel compositions are useful in creating valuable research tools, forexample antibodies against a composition known to result in the neuronalcell death associated with Alzheimer's Disease. The novel compositionsof the present invention can be used to create such antibodies byimmunizing an animal with these compositions and then isolating theresulting antibodies using methods known to those skilled in the art.These novel compositions are also useful for animal studies, for exampleby implanting the novel compositions into the brains of animal models todetermine under what conditions the symptoms of Alzheimer's disease canbe induced and then alleviated. Such studies are particularly usefulwhen performed with an additional experiment, namely simultaneouslyimplanting in the brain of a second animal a composition resulting fromadmixing Aβ peptide, one or ore promoting factors, and a compound whichsuppresses the formation of neurotoxic Aβ filaments in vitro. Lesssevere symptoms of Alzheimer's disease in the second animal comparedwith the first animal indicates that the compound can similarly suppressthe formation of neurotoxic Aβ filaments in vivo.

The methods of suppressing the formation of neurotoxic Aβ filaments andneuronal cell death have other uses when performed in vitro. Forexample, this method can be used to determine which interactions betweenAβ and the promoting factor are required for formation of a complexwhich is neurotoxic. In this method, the region(s) of Aβ and theregion(s) of the promoting factor which interact and result inneurotoxic filament formation are identified. The interactions which aredisrupted by the presence of the compound which suppresses the formationof neurotoxic filaments are also identified. This determination can bemade by techniques known to those skilled in the art, for example byx-ray crystallographic or NMR spectroscopic analysis of the complexesformed by Aβ peptide and the promoting factor both in the presence andin the absence of the compound which suppresses the formation ofneurotoxic filaments. This determination will facilitate the design anddiscovery of other agents which can be used to treat Alzheimer'sdisease.

The methods of the present invention can also be used to therapeuticallytreat an individual with Alzheimer's disease, i.e. to suppress theformation of neurotoxic Aβ filaments in the brain of an Alzheimerpatient or slow neuronal cell death in an Alzheimer patient. Treating anindividual with Alzheimer's disease by one of the methods of the presentinvention comprises administering a compound which is capable ofsuppressing the formation of neurotoxic filaments or slowing neuronalcell death to the individual so that a therapeutically effective amountof the compound contacts the requisite proteins and cells in the brainof the individual. These methods can also be used to treat an individualdiagnosed with Alzheimer's disease before the onset of symptomscharacteristic of the disease (e.g., loss of memory), thereby slowingthe onset of these symptoms. An individual can be diagnosed as havingAlzheimer's disease before the onset of symptoms characteristic ofAlzheimer's disease, for example, by methods disclosed in U.S. patentapplication Ser. No. 08/109,746, and U.S. Pat. No. 5,297,562, theteachings of which are hereby expressly incorporated into thisapplication. The compounds are administered, using known methods,including directly into the brain or into the ventricles of the brainthrough slow release from microcarriers, gels or chambers containingcells that are genetically engineered to produce the compound beingadministered. The compounds can be modified in a number of ways, asdiscussed above, to allow them to cross the blood-brain barrier, makingit possible to administer them orally, or by parenteral routes (e.g.,intramuscular, intravenous or subcutaneous). The form in which thecompounds are administered will be determined by the route ofadministration. Such forms include, but are not limited to, capsular andtablet formulations (for oral administration), liquid formulations (fororal, intravenous, intramuscular or subcutaneous administration) andslow releasing microcarriers, gels and chambers containing geneticallyengineered cells that produce the compound (for administrationintramuscularly, intravenously or into the brain ventricles). Theformulations can also contain a physiologically acceptable vehicle andoptional adjuvants, flavorings, colorants and reservatives. Suitablephysiologically acceptable vehicles may include saline, sterile water,Ringer's solution, and isotonic sodium chloride solutions. The specificdosage level of active ingredient will depend upon a number of factors,including biological activity of the particular preparation and age,body weight, sex and general health of the individual being treated.These compounds can also be administered to an individual withAlzheimer's disease in combination with other known treatments.

Other proteins are known to be associated with mature amyloid plaquesfound in the brains of Alzheimer's patients. Such proteins includeamyloid P component, heparin sulfate proteoglycan, complement proteinsand laminin protein (Eikelenboom and Stam, Acta Neuropathol. 57:239(1982), Coria et al., Lab. Invest. 58:454 (1988) and Snow et al., Am. J.Pathol. 133:456 (1988). In light of the discoveries disclosed herein, itis reasonable to assume that some or all of these proteins similarlypromote the formation of Aβ filaments and result in these filamentsbeing neurotoxic. It is also reasonable to assume that these proteinsare over-expressed by certain cells in areas of the brain that aresusceptible to forming determined by contacting these proteins with Aβunder conditions disclosed herein and following the rate of formation offilaments by the electron microscopy or light scattering experimentswhether any of these proteins are, in fact, promoting factors. It can bereadily ascertained whether these proteins are contributors to theneurodegenerative qualities of Aβ filaments by contacting these proteinswith Aβ in a culture of human cortical neurons or neuron-like PC-12cells and determining whether neuronal cell death is increased incomparison with cell death in cultures treated in the same manner butwithout the amyloid-associated protein being screened. Proteins whichare determined to be promoting factors and which enhance neurotoxicityare targets for therapeutic drugs in the treatment of Alzheimer'sdisease and can be used in assays for identifying such drugs. Suchmethods of treatment and assays are within the scope of the presentinvention.

Following a local inflammatory response or an injury, a number ofsystemic changes occur which, together, are called the acute phaseresponse (Kushner, Ann. N.Y. Acad. Sci. 389:39 (1982)). Some of theimportant manifestations of an acute phase response are fever, increasedrate of synthesis of a number of hormones, including glucocorticoids,and a rise in the concentration of a number of plasma proteins,including α₁ -ACT (Kushner, Ann. N.Y. Acad. Sci. 389:39 (1982), Bauer,et al., J. Biol. Chem. 97:866 (1983) and (Baumann et al., J. Immun.,139:4122 (1987)). The acute phase response can be induced by a number ofpurified hormones and/or defined secondary messenger analogs, includinginterleukin 1 (IL-1), interleukin 6 (IL-6), glucocorticoid anddexamethasone. Agents which can induce this acute phase response arereferred to herein as acute phase inducing agents. Results presentedherein show that dexamethasone acts synergistically with IL-1 toincrease the expression of α₁ -ACT in astrocytes. Dexamethasone andother acute phase inducing agents can therefore be used either alone orin conjunction with IL-1 in the methods of screening for a compoundwhich suppresses the formation of neurotoxic β-peptide filaments presentin an individual with Alzheimer's disease. These results suggest thatother acute phase inducing agents can similarly be used in this methodof screening. Which acute phase inducing agents are suitable can beidentified by the methods of the present invention, i.e. by determiningwhether α₁ -ACT mRNA is over-expressed in subconfluent cultures ofastrocytes in the presence of the acute phase inducing agent beingassessed.

The invention is further illustrated by the following examples, whichare not intended to be limiting in any way.

EXAMPLE 1

The Alzheimer Aβ Peptide Forms Amyloid Filaments in Vitro

Biosynthetic Aβ₁₋₄₂ was incubated at a concentration of 80 μM in 100 μlof 10 μM Tris- HCl; pH 7.0 at 22° C. for 8, 24 and 48 hours. Assembly ofAβ into high molecular weight filamentous structures was monitored bylight scattering at 400 nM. Light scattering increases linearlyaccording to the size and number of filaments formed. The results areshown in FIG. 1 and indicate that Aβ peptide spontaneously forms amyloidfilaments, starting after about 8 hours of incubation; the rate offormation levels off after about 24 hours.

At 18 hours, filaments were applied to carbon-coated Formvar on 200 meshcopper grids, dried, negatively stained with 3% uranyl acetate andvisualized in a JEOL10OCX electron microscope. Many small and a fewlarge filaments were visible in the electron micrograph and arerepresentative of the products of this polymerization reaction.

EXAMPLE 2

The Alzheimer Amyloid Associated Protein α₁ -ACT Promotes the Assemblyof Aβ Protein into Filaments

The Aβ₁₋₄₂ was incubated, as described in Example 1, in the presence orabsence of α₁ -antichymotrypsin or a control protein (BSA) at a molarratio of 200:1. Filament assembly was monitored by light scattering. Theresults are shown in FIG. 2 and indicate that α₁ -ACT results in therate of filament formation being enhanced about ten-37fold over thecontrols. Formation of filaments was essentially complete after about 8hours.

The degree of filament formation was analyzed using electron microscopy.The reaction mixture was diluted with water or buffer. A Formvar-coatedcopper electron microscope grid was touched briefly to a droplet of thereaction mixture, allowing the filaments to stick to the Formvar plasticfilm. This procedure was also performed with Formvar which was precoatedwith carbon prior to the sticking of the filaments to the surface. Thefilaments were then stained with either a solution of uranyl acetate orsprayed ("shadowed") with a heavy metal such as platinum to allow theirvisualization. Electron microscopic examination of the reaction productrevealed large numbers of long, 6 nM-wide, amyloid-like filaments, andmany shorter filaments. α₁ -ACT promoted filament formation at leastten-fold (see also FIG. 3).

EXAMPLE 3

The Amyloid Associated Protein Apolipoprotein E Promotes the Assembly ofAβ Peptide into Filaments

The Aβ₁₋₄₂ peptide was incubated in the presence of purifiedApolipoproteins E2, E3 and E4 and α₁ -ACT, ApoAI and ApoAII at a molarratio of 4:1, as described in Examples 1 and 2. Electron microscopicexamination of the reaction products was used to quantitate the amountAβ filament formation from the reaction of Aβ with ApoE3, ApoE4 or α₁-ACT. Five randomly chosen electron micrographs of each reaction productafter 18 hours of incubation as in Examples 1 and 2 were analyzed bycounting the number of crossovers that the filaments underwent with eachother. This provides a measure of both the number and length of theamyloid filaments per unit area of the grid. The results, shown in FIG.3, indicated that ApoE4, ApoE2, α₁ -ACT and ApoE3 all promote theformation of Aβ filaments. However, ApoE4 was about four times moreactive than ApoE3 or α₁ -ACT. ApoE2 was the least active of the ApoEproteins, promoting the formation of less than half the quantity of Aβfilaments than were produced in the presence of ApoE3 or α₁ -ACT. Theformation of Aβ filaments was inhibited when ApoE4 was incubated with Aβin the presence of ApoE2 and equimolar amounts of ApoE4 and ApoE2 wereused. Reducing the amount of Aβ filament formed to levels produced bythe control protein, BSA (see FIG. 3). ApoA1, ApoAII and BSA wereinactive.

EXAMPLE 4

Neurotoxicity of Alzheimer β-Peptide Filaments Formed in the Presence ofPromoting Factors

Certain promoters of the assembly of Aβ filaments of amyloid filamentsformed in vitro also result in these filaments being neurotoxic. Thiswas determined by using fetal human cortical tissue from 18-22 weekgestation were used for this study. The protocol obtaining postmortemfetal tissue complied with all federal guidelines for federal research.The cortical cell culture were prepared as follows. The frontal andtemporal lobes were removed from fetal brain. Cells were disassociatedin calcium and magnesium free Hank's balanced salt solution containing0.2% tyrosine for 10 minutes at room temperature, and subsequentlydisassociated in serum-containing Dulbecco's Modified Eagle Medium(D-MEM) with DNase (0.2 mg/ml) for three times. The cells were platedonto laminin (10 μg/ml) coated 96-well plate (10⁻⁴ cell/well) in aneuronbasal medium supplemented with B27 (Gibco), 2 mM glutamine and 1×antibiotic antimycotic solution and kept in a humidified 5% CO₂atmosphere. After 2-3 days in vitro (DIV), non-neuronal cell divisionwas halted by 2 days exposure of 10⁵ cytosine arabinoside and cells wereshifted into the plating medium. Subsequent media replacement wascarried out on a biweekly schedule.

The products of the reactions shown in FIG. 3 were bath applied to twoor three sister cultured wells on day 12. Cell viability was evaluatedby morphological criteria and calorimetric MTT (Tetrazolium) on day 16.MTT 5 mg/ml was dissolved in serum free D-MEM and filtered to sterilize.25 μl of the 5 mg/ml solution of MTT was applied to each assayed wellsfollowing removal of all culture medium from the plates, and plates wereincubated at 37° C. for 3 hours. 100 μl dimethyl sulfoxide (DMSO) werethen added into each well and mixed thoroughly to dissolve the darkcrystals. The plates were read on a BT 1000 microreader, using a testwavelength of 550 nm, a reference wavelength of 650 nM. The plates wereroutinely read within 20 minutes of addition of DMSO.

The results are shown in FIG. 4. The greatest neurotoxicity wasexhibited by the reaction mixtures containing Aβ plus α₁ -ACT or Aβ plusApoE4. Aβ alone was also neurotoxic, possibly by reacting first withα-ACT-like proteins in the serum used to grow the cells. ApoE3 not onlydid not promote the development of neurotoxic activity, but reduced theactivity of the Aβ peptide itself.

Similar results were also obtained using neuron-like PC-12 cells andneonatal rat cortical neurons as targets for the neurotoxic activity ofamyloid filaments. The procedure for performing the assay forneurotoxicity using neuron-like PC-12 cells is the same as described isExample 5 except that the blocking peptides are not added to the assay.

EXAMPLE 5

The Effect of Blocking Peptides on the Neurotoxicity of Alzheimerβ-Peptide Filaments

PC-12 cells were grown on normal tissue culture plastic inserum-containing medium. When they reached 75% confluence, they werereplated in two sets of 96-well dishes precoated with poly-D-lysine andlaminin at 10⁶ cells per well in N2 medium. After two days, NGF at 100ng/ml was added to each well to induce neurite outgrowth. 1-2 dayslater, the cells were fed with fresh N2 medium lacking NGF. The reactionmixtures containing β-protein, ±α₁ -antichymotrypsin, Apolipoprotein E,or the various blocking peptides, were added one day later. Reactionmixtures containing β-protein, +α₁ -antichymotrypsin and the variousblocking proteins along with reaction mixtures containing appropriatecombinations thereof for controls were added to the first set of dishes.Reaction mixtures containing β-protein, Apolipoprotein E4 and thevarious blocking proteins along with reaction mixtures containingappropriate combinations thereof for controls were added to the secondset of dishes. After one more day of incubation, the MTT release assaywas carried out to measure neurotoxicity.

FIG. 5 shows the results of the set of dishes which tested the abilityof blocking proteins to suppress the neurotoxicity of β-protein and α₁-antichymotrypsin. Reaction mixtures containing only β-protein and α₁-antichymotrypsin were highly neurotoxic. However, reaction mixturescontaining β-protein, α₁ -antichymotrypsin and Aβ₁₋₁₂ were about fivefold less neurotoxic. In the data shown, standard errors of the means ofseparate wells in the 96-well dish are presented.

FIG. 6 shows the results of the set of dishes which tested the abilityof blocking proteins to suppress the neurotoxicity of β-protein andApolipoprotein E4. Reaction mixtures containing only β-protein andApolipoprotein E4 were highly neurotoxic. However, reaction mixturescontaining β-protein, α₁ -antichymotrypsin and Aβ₁₂₋₂₈ were about fourto five fold less neurotoxic. In the data shown, standard errors of themeans of separate wells in the 96-well dish are presented.

EXAMPLE 6

Determination of the Level of ACT mRNA in Fetal Human Brain

Mixed glial cultures were prepared from portions of human brain of20-week gestational age from which the meninges, mid, and hind brainshad been removed. The tissue was then homogenized and treated withtrypsin for minutes at 37° C. A portion of the cells were set aside.Total RNA was isolated from these cells, as described below. Theremainder of the partially dissociated cells were suspended in DMEMsupplemented with 10% fetal calf serum, 1 mM pyruvate, 100 U/mlstreptomycin-amphotericin-penicillin (Sigma, Hybrimax), and 2 mMglutamate. They were triturated 20-25 times and plated on 20 cm plates.The cells were grown in the serum-containing medium until they reachedconfluence (about 1 week).

After one-two weeks, the majority of neurons had died and detached fromthe substrate, leaving a confluent monolayer of glial cells. From themorphology of the cells, it appeared that most were astrocytes, togetherwith a few microglia. This conclusion was confirmed by immunolabeling ofthe cells with an antibody to the astrocyte specific protein glialfibrillary acidic protein (GFAP). Cells to be stained for GFAP weregrown on poly L-lysine coated coverslips, fixed for 5 minutes in 4%paraformaldehyde at room temperature, then permeabilized with 0.1%Triton X-100 for 1 hour at room temperature. Coverslips with cells werethen incubated with a 1/400 dilution of the primary antibody (mousemonoclonal antiGFAP, Sigma Chemicals) at 4° C. overnight. They were thenwashed three times for 5 minutes each with PBS. The coverslips were thenincubated with the secondary antibody (goat-anti-mouse TRITC conjugate,1/500 dilution, Sigma Chemicals) for 1 hour at room temperature, washedthree times, and mounted on slides for visualization and photography.Visualization was by means of a Zeiss fluorescence photomicroscope.GFAP-positive cells are identified by the fluorescence labeling of theirinternal filamentous cytoskeleton. The results indicated that >90% ofthe cells were indeed astrocytes.

One plate of astrocytes was used in each experimental test. Completemedium was first replaced with serum-free N2 medium (high glucose DMEM,2 mM glutamate, 100U/ml strep-amp, 1 mM pyruvate, 5 μg/ml insulin, 100μg/ml transferrin, 100 μM putrescine, 30 nM selenium, and 20 nMprogesterone) for 2 hours. The cells were then exposed to the differenttreatments for 6 hours, except where noted. Following treatment, thecells were harvested, and their RNA isolated for Northern blot analysis.

Total cellular RNA was isolated as described by Chirgwin et al. (1979).Briefly, cells (-10 mg of tissue) were washed with phosphate-bufferedsaline (PBS) and lysed directly on the culture plate in 1 ml of 4 Mguanidinium isothiocyanate/25 mM sodium citrate, pH 7/0.5% sarcosyl/0.1M2-mercaptoethanol. The cell lysate was layered on top of 2 ml of 5.7Mcesium chloride and centrifuged overnight at 39,000×g. The RNA pelletobtained was resuspended in water, ethanol precipitated, resolubilizedin water, and quantitated using spectrophotometry. 3-10 μg samples ofRNA were denatured in 2.2M formaldehyde/50% formamide (vol/vol) andsubjected to electrophoresis in a 1% agarose gel containing 2.2Mformaldehyde. The gels were stained with ethidium bromide andphotographed to verify that each lane contained equal amounts ofundegraded RNA. After transfer to nitrocellulose, the RNA was hybridizedto ³² p!-labeled probe. Probes were labeled by priming with randomhexamer oligonucleotides in the presence of ³² p!-dCTP (New EnglandNuclear). After hybridization, the membranes were washed underconditions of high stringency (0.1X SSC, 55° C.), and exposed to XAR-5autoradiography film (Kodak). ACT MRNA was undetectable in fetal humanbrain. However, the autoradiogram indicated a high level of ACT mRNA inserum grown cells that had reached confluence.

EXAMPLE 7

Induction of ACT in Human Astrocytes by IL-1

Mixed glial cultures from human fetal frontal cortex, cerebellum, andbrain stem were prepared as described in Example 6, grown tosubconfluence in serum-containing medium, then switched to serum-free N2medium for 2 hours. The cultures were then treated with IL-1 (250 U/ml)and/or dexamethasone (1 μm) for 4 hours, at which time RNA was isolatedand subjected to Northern blot analysis with an ACT probe as describedin Example 6. IL-1 alone was able to induce ACT expression in cells fromthree brain areas. There is a synergistic effect of adding dexamethasonetogether with IL-1. The main difference between the different culturesis that cells prepared from the cortex have a much higher level ofendogenous ACT mRNA compared to cells prepared from the cerebellum orbrain stem.

EXAMPLE 8

Constitutive Expression of ACT in Cortical but not Cerebellar or BrainStem Mixed Glial Cultures

Cultures of mixed glial cells were prepared as described in Example 6from human fetal frontal cortex, cerebellum, and brain stem, grown forone to two weeks in serum-containing medium to approximately 80%confluence.

The cultures were then switched to N2 medium and allowed to reachconfluence over the ensuing twelve hours. RNA was prepared and subjectedto Northern blot analysis using an ACT probe as described in Example 6.Cultures prepared from the frontal cortex expressed a high spontaneouslevel of ACT mRNA, which could not be further induced by the addition ofIL-1 and dexamethasone. In contrast, the cultures prepared from thecerebellum or brain stem at various times after they reached confluencecontinued to express the low level of ACT mRNA seen in subconfluentcultures and were again sensitive to induction by IL-1 anddexamethasone.

EXAMPLE 9

Blockade of Constitutive ACT mRNA Expression in Cortical Mixed GlialCultures by Antibody to the IL-1 Receptor

Human cortical mixed glial cultures were prepared and allowed to reachconfluence as described in Examples 6-8, either in the absence orpresence of 2.5μg/ml blocking antibody to the IL-1 receptor. Suitableblocking antibodies to the IL-1 receptor are commercially available fromGenzyme Corporation, or can be obtained by immunizing a mouse with IL-1receptor protein, immortalizing the resulting isolated spleen cells byfusion with a hydridoma partner, and assaying for expressed antibodieswhich block the IL-1 receptor.

The RNA of the cells of the two cultures was then subjected to Northernblot analysis. Total cellular RNA was isolated as described by Chirgwinet al., Biochemistry, 18:5294-5299 (1979). Cells (about 10 mg of tissue)were washed with phosphate-buffered saline (PBS) and lysed directly onthe culture plate in 1 ml of 4 M guanidinium isothiocyanate/25 mM sodiumcitrate, pH 7/0.50% sarcosyl/0.1M 2-mercaptoethanol. The cell lysate waslayered on top of 2 ml of 5.7M cesium chloride and centrifuged overnightat 39,000×g. The RNA pellet obtained was resuspended in water, ethanolprecipitated, resolubilized in water, and quantitated usingspectrophotometry. 3-10 μg samples of RNA were denatured in 2.2Mformaldehyde/50% formamide (vol/vol) and subjected to electrophoresis ina 1% agarose gel containing 2.2M formaldehyde. The gels were stainedwith ethidium bromide and photographed to verify that each lanecontained equal amounts of undegraded RNA. After transfer tonitrocellulose, the RNA was hybridized to ³² p!-labeled probe derivedfrom either the human ACT cDNA or the rat ACT homologue, contrapsin,cDNA. Probes were labeled by priming with random hexameroligonucleotides in the presence of ³² p!-dCTP (New England Nuclear).After hybridization, the membranes were washed under conditions of highstringency (0.1X SSC, 55° C.), and exposed to XAR-5 autoradiography film(Kodak). Experiments were repeated at least 3 times with similarresults.

Northern blot analysis of equal amounts of total RNA isolated from thetwo cultures indicated that the spontaneous level of ACT mRNA wasreduced (at least five-fold) in the presence of the antibody.

EXAMPLE 10

Removal of Microglial Cells from Mixed Cultures and Quantitation of α₁-ACT Produced by the Remaining Astrocytes

Mixed glial cultures from human fetal front cortex, cerebellum and brainstem were prepared as described in Example 6. Pure astrocytes wereprepared by 3 different protocols: (1) Plated cells were incubated with0.2 mg/ml trypsin for 15 minutes. The supernatant, including nonadherentcells, was discarded. The remaining cells were then washed with HBSS andplaced in fresh medium. (2) A standard procedure for obtaining pureastrocyte cultures was carried out according to (Lee, et al., LabInvest, 30 67:465 (1992)). The subconfluent mixed glial cultures wereplaced in a rotary shaker at 180 rpm overnight. The supernatant andnon-adherent cells were discarded and the remaining cells washed andsupplemented with fresh medium. (3) Cultures were treated with 5 mMH-Leu-O-Methyl ester for 18 hours according to (Giullian, J. Jeurosci.Res., 18:155 (1987)), after which the adherent cells were washed andfresh medium added. In all three procedures, the cells remainingwere >95% astrocytes as determined by morphology. The number ofmicroglia removed from (and therefore present in) the mixed glialcultures was assessed by staining with two macrophage/microglialcellspecific antisera, LN3 and Macl (ICN) and were found to be equalfrom all brain regions ±10-15%.

One plate of mixed glial or pure astrocytes was used in eachexperimental test. Complete medium was first replaced with serum-free N2medium (high glucose DMEM, 2 mM glutamate, 100U/ml strep-amp, 1 mMpyruvate, 5 μg/ml insulin, 100 μg/ml transferrin, 100 μM putrescine, 30nM selenium, and 20 nM progesterone) for 2 hours. The cells were thenexposed to IL-1 (250 U/ml) and/or dexamethasone (1 mm) for 6 hours.Following treatment, the cells were harvested, and their RNA isolatedfor Northern blot analysis, as described in Example 6. Pure astrocytesshowed no difference from mixed glia cultures in the amount of α₁ -ACTproduced in the presence of IL-1 and/or dexamethasone.

EXAMPLE 11

Quantitation of IL-1 Positive Cells from Different Areas of AlzheimerBrain

Immunohistochemistry for IL-1 was performed as described by (Griffin, etal., Proc Natl. Acad. Sci. USA 86:7611 (1989)). Briefly, paraffin-fixedsections from the cerebellum and hippocampus regions from AlzheimerDisease and control brain were processed on slides through three changesof xylenes and rehydrated by processing slides through 100%, 95%, and75% ethanol. Sections were permeabilized in 0.05% Triton X-100 for 10minutes followed by 0.2 N HCl for 20 minutes. Endogenous peroxidase wasblocked by placing sections in 0.6% H₂ O₂ /methanol for 30 minutes.Blocking of sections was performed in 20% goat serum for 30 minutes.Appropriate dilution of the primary antibody (1:20 for anti-human 1L-1α(Ciston), or direct application of monoclonal LN3 (ICN) was then appliedto the sections overnight at 4° C. For 1L-1α staining, a PAP procedurewas used. 1:50 dilution of goat anti-rabbit (Sigma) IgG was applied totissue sections for 30 minutes. 1:300 dilution of rabbit peroxidaseanti-peroxidase was then applied for 30 minutes to the section. Finallythe sections were developed with DAB, counter-stained with hematoxylin,dehydrated and mounted with coverslips. For LN3 staining, the Vector ABCVectastain kit was used for peroxidase staining, following whichsections were counter-stained with hematoxylin, dehydrated and mountedwith slipcovers.

The staining, i.e., IL-1 positive glial cells, are not increased to thesame extent in the cerebellum as in the hippocampus region of the brainof Alzheimer patients.

EXAMPLE 12

Biosynthetic Aβ1-42 (80 μM) was incubated with ACT (CalBiochem),purified human apoE and individual control protein (0.4 μM) in 100 μl of10 μM Tris-HCl, pH 7.0 for 48 hours at 22° C. The 12.5 μl of eachreaction product was then added to 14 days old primary human corticalneurons in culture. The cell viability was measured by a calorimetricMTT (Sigma) assay on day 4. Data are expressed ± S.E.M. in FIGS. 7-9.ACT and apoE4 induced a 2- and 4-fold increase in neurotoxicity ofAβ1-42, respectively. ApoE2 had a slight effect on Aβ1-42 neurotoxicity.Since Aβ1-42 is prone to self-aggregate into filaments, only aliquots ofeach preparation of Aβ1-42 devoid of filaments, as judged by electronmicroscopy, were used for experiments.

Method of cell culture: The cortical neuron cultures were prepared asfollows. Frontal lobes of brain were removed from an electivelyterminated human fetus on gestational age of 18-21 weeks. Cortical cellswere dissociated in serum-free medium containing 0.2% trypsin (Sigma)and 25 μg/ml DNase I (Sigma) and plated 4×10⁴ cells per well (96-wellplate, Falcon) precoated with 10 μg/ml laminin (Gibco). The cells werecultured in DMEM (Gibco) supplemented with 10% fetal calf serum (Gibco)and 10 2 mM glutamine. Cultures were kept at 37° C. in a 7% CO₂atomsphere. After day 4 in vitro, non-neuronal cell division was haltedby exposure to 10⁻⁵ M cytosine arabinoside. Culture medium was changedevery 3-4 days with complete medium, and changed to neurobasal mediumcontaining B27 (Gibco) one day before addition of each reaction product.

Chemically synthesized Aβ2-9 (0.8 μM) or Aβ12-28 (0.8 μM) waspreincubated with ACT (0.4 μM) or apoE (0.4 μM) for 2 hours followed byaddition of Aβ1-42 (80 μM). The conditions of incubation and the MTTassay were as described earlier in this Example. Aβ2-9 reduced ACTactivity in promoting the formation of neurotoxic Aβ1-42, 2-2.4-foldAβ12-28 and apoE2 decreased apoE4 function in promoting the formation ofneurotoxic Aβ1-42 6-9 fold.

The experimental procedure is as described as above in this Example.Each reaction product was applied to carbon coated Formvar on 200 meshcopped grids, washed and negatively stained with 1% uranyl acetate andvisualized in a JEOL-100CS electron microscope. The indicatedconcentrations of Aβ2-9, Aβ12-28 or apoE2 were incubated with ACT orapoE4 following addition of Aβ1-42 as described earlier in this Example.The reduction of ACT neurotoxic Aβ1-42 promoting activity by Ag2-9 wasdetected at 1 nM concentration. The reduction of apoE4 neurotoxic Aβ1-42promoting activity by Aβ12-28 or apoE2 was observed at 1 nM or 10 nMconcentration, respectively.

Equivalents

Those skilled in the art will know, or be able to ascertain using nomore than routine experimentation, many equivalents to the specificembodiments of the invention described herein. These and all otherequivalents are intended to be encompassed by the following claims.

What is claimed is:
 1. An oligopeptide, wherein the oligopeptide isselected from the group consisting of Aβ₂₋₉, a fragment of Aβ₂₋₉ or amodified fragment of Aβ₂₋₉, wherein the oligopeptide binds α₁-antichymotrypsin.
 2. An oligopeptide, wherein the oligopeptide is Aβ₂₋₉or a fragment thereof, wherein the oligopeptide binds α₁-antichymotrypsin.
 3. The oligopeptide of claim 2, wherein theoligopeptide is Aβ₂₋₉.
 4. An oligopeptide, wherein the oligopeptide is afragment of Aβ₁₋₁₂, or a modified-fragment of Aβ₁₋₁₂, wherein theoligopeptide binds α₁ -antichymotrypsin, with the proviso that theoligopeptide is not Aβ₁₋₁₀.
 5. The oligopeptide of claim 4 wherein theoligopeptide is Aβ₂₋₁₀, Aβ₂₋₁₁, Aβ₁₋₁₁, or Aβ₁₋₉.
 6. An oligopeptide,wherein the oligopeptide is a fragment of Aβ₁₂₋₂₈ or a modified fragmentof Aβ₁₂₋₂₈, wherein the oligopeptide binds ApoE4, with the proviso thatthe oligopeptide is not Aβ₁₇₋₂₀ or Aβ₁₈₋₂₈.